Project Summary Colorectal cancer is the second leading cause of cancer death in the United States. As current treatments only show limited effectiveness, there is a critical need for new treatment strategies. Previous studies demonstrated that (sub)telomere maintenance contributes to cancer formation, likely due to its underlying role in genomic instability. In this proposal, we aim to study how (sub)telomere dysfunction arises and to identify key genomic and epigenetic instabilities that occur at early stages of cancer development using a well-established carcinogenic progression model of human colorectal cancer. We hypothesize that genetic and epigenetic variations in telomere dysfunction arise in a chromosome-arm specific manner and are associated with molecular subtype and malignancy of colorectal cancer. Aim 1 will test the cooperative regulation of telomere maintenance machinery by subtelomere elements in cultured human cells. We will employ CRISPR/Cas9 techniques in the cells to disrupt subtelomere elements in order to determine its structural effect on telomere maintenance machinery. In addition, we will determine molecular interactions in telomere dysfunction mediated by crosstalk effects between unbalanced DNA methylation and DNA replication stress. Impact: This aim will identify currently unknown structural and epigenetic mechanisms of (sub)telomere maintenance associated with early colorectal cancer. Aim 2 will determine the extent and the nature of end-to-end chromosome fusion in human biopsies isolated from histologically defined sites of colorectal tissue. Recently, we developed a TAR- fusion PCR assay, an original and proven technique to detect telomere fusions in solid tissue DNA. We will employ this technique to determine the frequency of telomere fusion using paired tumor and adjacent tissue specimens and will utilize DNA sequence analysis to identify the key internal structures of telomere fusion junctions. Impact: The identification of key DNA sequences of telomere fusion junctions associated with malignancy will provide novel targets for cancer detection and prevention. Our proposed research will examine properties of cells that can be used to identify and target the development of future tumors that arise during the early stages of colorectal cancer and to further elucidate fundamental questions regarding the role of telomere dysfunction in early colorectal carcinogenesis. These studies are likely to open new avenues for colorectal cancer research and lead to earlier detection and improved outcomes for patients with colorectal cancer.